Untitled Flashcards Set

Pipetting

The technique of using a pipette to accurately measure and transfer liquid.

Primary structure

The sequence of amino acids in a protein.

Active site

The region on an enzyme where substrate binding occurs and the chemical reaction takes place.

Ion-exchange chromatography

A technique for separating proteins based on their charge.

SDS-PAGE

A method used to separate proteins based on their size by using sodium dodecyl sulfate.

PCR (Polymerase Chain Reaction)

A technique used to amplify a specific DNA sequence.

Agarose gel

A medium used in gel electrophoresis to separate DNA or RNA fragments by size.

Hermaphrodite (in C. elegans)

An organism that has both male and female reproductive organs.

Chemotaxis

The movement of an organism in response to a chemical stimulus.

Model organism

A non-human species that is studied to understand biological processes.

Wildtype

The typical form of a species or strain in nature, as opposed to mutants.

Mutant

An organism that has undergone a mutation, resulting in a variant form of the gene.

Substrate

The reactant molecule upon which an enzyme acts.

Activation energy

The minimum amount of energy required to initiate a chemical reaction.

Alpha helix

A right-handed coiled conformation of proteins, often involved in secondary structure.

Beta pleated sheet

A common motif in the secondary structure of proteins where strands are arranged side by side.

N-terminus

The start of a protein or polypeptide chain, characterized by a free amino group.

C-terminus

The end of a protein or polypeptide chain, characterized by a free carboxylic acid group.

Hydrophobic amino acids

Amino acids that do not interact well with water and are typically found in the interior of proteins.

Hydrophilic amino acids

Amino acids that are attracted to water and are often found on the surface of proteins.

For step 1 of chromatography, What proteins would you expect to elute from the column upon adding the equilibration buffer?

None. You haven’t added any protein to the column yet, so no protein would elute at this step (trick question)

Add the solution containing your protein of interest to the column and collect the flow through.

In our case the solution we added to the column was egg white diluted in 25 mM glycine buffer (pH 9.2).

Q: What charges would you expect the proteins that elute at this step to have?

The proteins that elute at this step would likely be positively charged due to the alkaline pH of the glycine buffer, which favors the elution of proteins with a net positive charge.

Wash out unbound proteins from the column using the equilibration buffer.

In our case the equilibration buffer was 25 mM glycine at pH 9.2.

Q: What charge(s) would you expect the proteins that elute at this

step to have?

Negative (repelled from negatively charged Sephadex beads) and neutral (not attracted to negatively charged Sephadex beads) proteins would elute at this step.

. Wash out weakly unbound proteins from the column using a low salt solution.

In our case the low salt solution was 100 mM NaCl in equilibration buffer (i.e., 25 mM glycine (pH 9.2)).

Q: What charge(s) would you expect the proteins that elute at this

step to have

Weakly positive

Elute your protein of interest using a high salt solution

In our case the high salt solution was 750 mM NaCl in equilibration buffer (i.e., 25 mM glycine (pH 9.2)).

Q. What charge(s) would you expect the proteins that elute at this

step to have?

A: Strongly positive

Be able to determine the concentration of protein in an unknown solution using a standard curve

Q: You generate a standard curve of BSA using a Bradford assay. The

equation of the curve is: y=0.02x (y-axis is absorbance, x-axis is ug of

BSA). You measure the absorbance of 100 uL of an unknown solution,

and the spectrophotometer reads 0.6 AU. What is the concentration of protein in the unknown solution?

A: 0.3 ug/uL Plug in y and solve for X

Q: How many uL of unknown sample will you need to load 15 ug of

protein in an SDS-PAGE well? The concentration of protein in the

unknown sample is 2 ug/uL

A: 7.5 uL

Q: What is PCR used for?

A: Amplifying a specific target sequence of DNA determined by the primers used. (i.e., Making lots of copies of DNA. The sequence that is copied is the region flanked by the forward and reverse primers.)

Q: What are the necessary components of PCR?

A: primers, polymerase, nucleotides, template DNA

Q: What are agarose gels used for?

A: Separating DNA (or RNA) by size

Q: Toward which electrode (+ or -) does DNA (or RNA) run on an agarose gel?

A: Towards the positive electrode because DNA (and RNA) is negatively charged.

Q: Do small or large DNA (or RNA) fragments move faster through an agarose gel?

A: Small DNA (or RNA) fragments move faster (large fragments move slower)